Novel lactobacillus paracasei strain

ABSTRACT

A major object of the present invention is to provide an effective means for promoting polyamine synthesis in an organism (in particular, in humans). 
       Lactobacillus paracasei  having polyamine production promoting activity in an organism.

TECHNICAL FIELD

The present invention relates to a novel Lactobacillus paracasei strain(strain WON0604: FERM BP-11468) having polyamine production promotingactivity in an organism, as well as its application and relevanttechnologies.

BACKGROUND ART

Polyamine is a general name for aliphatic hydrocarbons having two ormore primary amino groups. Typical examples of polyamine includeputrescine, spermidine, spermine, and the like. Polyamine is synthesizedin the cells of all organisms, and is involved in cell differentiationor proliferation. In addition to these activities, there are recentreports that polyamine has various helpful physiological activities,including an anti-aging effect (Non-patent Document 1), arteriosclerosisprogression retardation activity (Non-patent Document 2), acute andchronic inflammation suppressing activity (Non-patent Document 3),neutral fat reducing activity, insulin resistance alleviating activity,anti-obesity activity, cholesterol level decreasing activity, basalmetabolism increasing activity (Non-patent Document 4), and antiallergicactivity (Non-patent Document 5).

Polyamine biosynthesis occurs in all animals, including humans; however,this synthesis ability decreases with aging. Therefore, in order toreceive the helpful physiological activity of polyamine over a lifetime,there have been discussions regarding external administration or intakeof polyamine, or activation of polyamine synthesis ability in anorganism.

To date, several food and drinks containing polyamine have beensuggested. For example, Patent Document 1 discloses food and drinkscontaining polyamine extracted from various plant/animal-originated rawmaterials. Such food and drinks are expected to promote polyaminesynthesis in organisms (in particular, in humans), and thereby providethe aforementioned various helpful physiological activities, such as ananti-aging effect, arteriosclerosis progression retardation activity,acute and chronic inflammation suppressing activity, neutral fatreducing activity, insulin resistance alleviating activity, anti-obesityactivity, cholesterol level decreasing activity, basal metabolismincreasing activity or antiallergic activity. Further, such food anddrinks are expected to act, for example, on the improvement, retainmentor homeostasis of biomarkers or the like relevant to the abovephysiological activities, or on prevention of development of diseases.

CITATION LIST Patent Documents

-   Patent Document 1: JP2010-263816A

Non-Patent Documents

-   Non-patent Document 1: Eisenberg T. et al., Nat Cell Biol 2009; 11    (11): 1305-1314-   Non-patent Document 2: Soda K., Med. Hypotheses, 2010; 75 (3):    299-301-   Non-patent Document 3: Lagishetty C.V. et al., Indian J. Pharmacol.    2008; 40 (3): 121-125-   Non-patent Document 4: Koponen T. et al., Amino Acids 2012; 42 (2-3)    : 427-40.-   Non-patent Document 5: Peulen 0. et al., Public Health Nutr. 1998; 1    (3): 181-184

SUMMARY OF INVENTION Technical Problem

An object to be attained by the present invention is to provide aneffective means for promoting polyamine synthesis in organisms (inparticular, in human organisms).

Solution to Problem

In an attempt to attain the above object, the inventors of the presentinvention found, among the microorganisms belonging to Lactobacillusparacasei, a microorganism capable of promoting polyamine production inhuman or animal organisms. The inventors of the present invention foundthat this microorganism has polyamine production promoting activity inthe small intestine, as well as hepatic neutral fat reducing activityand/or energy metabolism promoting activity.

Representative examples of the present invention are detailed below.

Item 1.

Lactobacillus paracasei strain WON0604 (FERM BP-11468).

Item 2.

Lactobacillus paracasei strain WON0604 according to Item 1, furtherhaving hepatic neutral fat reducing activity.

Item 3.

Lactobacillus paracasei strain WON0604 according to Item 1 or 2, furtherhaving energy metabolism promoting activity.

Item 4.

A composition to which Lactobacillus paracasei strain WON0604 accordingto any one of Items 1 to 3 has been added.

Item 5.

The composition according to Item 4, wherein the composition is apolyamine production promoter.

Item 6.

An agent for preventing or treating at least one disease selected fromthe group consisting of fatty liver, non-alcoholic steatohepatitis, andliver cirrhosis, the agent comprising the Lactobacillus paracaseiaccording to Item 2.

Item 7.

An agent for reducing hepatic neutral fat, comprising the Lactobacillusparacasei according to Item 2.

Item 8.

A method for promoting polyamine production in a human in need ofpolyamine production enhancement, comprising administering Lactobacillusparacasei strain WON0604 according to any one of Items 1 to 3 to thehuman.

Item 9.

A method for treating or alleviating a fatty liver patient, comprisingadministering Lactobacillus paracasei strain WON0604 according to Item 2to the fatty liver patient.

Item 10.

Use of the Lactobacillus paracasei according to Item 2 for themanufacture of an agent for preventing and/or treating fatty liver.

Advantageous Effects of Invention

Lactobacillus paracasei strain WON0604 (FERM BP-11468; this strain mayhereinafter also be referred to as “the microorganism of the presentinvention”) of the present invention has polyamine production promotingactivity in organisms of human or other animals. In the presentinvention, this activity is referred to as “polyamine productionpromoting activity.” By using the microorganism of the present invention(for example, through ingestion or administration), it is possible topromote polyamine production in a human or animal organism.Consequently, the invention enables a human or an animal to effectivelyreceive helpful physiological activities of polyamine (for example, ananti-aging effect, arteriosclerosis progression retardation activity,acute and chronic inflammation suppressing activity, neutral fatreducing activity, insulin resistance alleviating activity, anti-obesityactivity, cholesterol level decreasing activity, basal metabolismincreasing activity, antiallergic activity, and/or immunostimulatoryactivity, etc.).

Further, the microorganism of the present invention has hepatic neutralfat reducing activity. Therefore, by using the microorganism of thepresent invention, it is possible to decrease the hepatic neutral fatlevel (for example, the amount of the neutral fat accumulated in theliver). The microorganism of the present invention is effective for theretainment or improvement of hepatic lipid metabolism, or prevention ortreatment of fatty liver, non-alcoholic steatohepatitis (NASH), and/orliver cirrhosis. Further, the microorganism of the present invention isexpected to have an effect of preventing the progression of thesediseases into liver cancers, or an effect of preventing the developmentof cardiovascular diseases.

The microorganism of the present invention has energy metabolismpromoting activity in an organism. Therefore, use of the microorganismof the present invention is effective for health maintenance of humansor animals, including suppression of body weight gain, suppression ofobesity, reduction in body fat, reduction in visceral fat, and/orprevention of metabolic syndrome. Further, the microorganism of thepresent invention is expected to have an effect on improvement orretainment of biomarkers relevant to the above physiological activities.

In addition, since the microorganism of the present invention belongs toLactobacillus paracasei, which have been applied in the food field, themicroorganism of the present invention is believed to be sufficientlysafe as an additive for food compositions. As is thus evident, themicroorganism of the present invention is suitable for the fields offood and drinks and/or pharmaceuticals for humans or other animals.

DESCRIPTION OF EMBODIMENTS 1. Microorganism

1-1. Polyamine production promoting activity

The microorganism of the present invention has polyamine productionpromoting activity. As described above, “polyamine production promotingactivity” is an action of promoting polyamine production in organisms ofhumans or other animals.

The “polyamine” used in the present invention is generally a collectivename for aliphatic hydrocarbons having two or more primary amino groups,which is a substance recognized as polyamine. Examples of polyamineinclude putrescine, spermidine and spermine. The polyamine productionpromoting activity of the microorganism of the present inventionpromotes production of at least one kind or two kinds, more preferablyall kinds of polyamine. The cells, tissues, and organs in whichpolyamine production (i.e., polyamine synthesis in the organism) ispromoted by the microorganism of the present invention are notparticularly limited. Examples of the organs include oral cavity,esophagus, stomach, duodenum, cecum, small intestine, and largeintestine, which are organs in which the microorganism orallyadministered directly takes effect. Small intestine is particularlypreferable.

The polyamine production promoting activity of the microorganism of thepresent invention can be measured using a known analysis technique.Specifically, the polyamine production promoting activity of themicroorganism of the present invention can be measured in the followingmanner using a model animal (for example, mice). More specifically, miceare fed with the microorganism of the present invention for a certainperiod of time, then their visceral tissues were isolated, and thepolyamine amounts in the tissues are measured. Further, the measuredpolyamine amounts are compared with the polyamine amounts in thevisceral tissues isolated from mice that have not been fed with themicroorganism of the present invention, thereby measuring a relativeincrease by the microorganism of the present invention. In thismeasurement, the sample used for the polyamine amount measurement is notparticularly limited. It is, however, preferable to measure and comparethe polyamine amounts in the small intestine tissue and the cecalcontent. For example, when the measurement reveals that the polyamineamount in the small intestine increased whereas the polyamine amount inthe cecum content did not increase, it can be judged that themicroorganism has an action of promoting polyamine production in anorganism.

It is preferable that the polyamine amount in the organism be increasedby a factor of 1.1 to 1.5 by the administration of the microorganism ofthe present invention to humans or other animals, compared with thatbefore the administration. This is, for example, to receive an effect ofreducing an increase in hepatic neutral fat by suppressing excessivelipid accumulation or promoting lipid metabolism in the organism.

1-2. Mycological Characteristics

The microorganism of the present invention belongs to Lactobacillusparacasei. Table 1 below shows preferable mycological properties of themicroorganism.

TABLE 1 Shape of cell Rod shape Gram staining Positive Motility NoneSpore None Growth temperature 30-40° C. Growth under anaerobic Positivecondition Growth under aerobic condition Positive Gas Production None

1-3. Characteristics Regarding Colony Formation

The microorganism of the present invention preferably forms colonieshaving the characteristics below when the microorganism is cultured inagar medium having the formulation below (Lactobacilli MRS Agar#288210)at 37° C. for 16 hours under an anaerobic condition.

Medium Formulation (pH 6.5±0.2)

Proteose Peptone No. 3 10 g Beef extract 10 g Yeast extract 5 g Glucose20 g Polysorbate 80 1 g Ammonium citrate 2 g Sodium acetate 5 gMagnesium sulfate 0.1 g Manganese sulfate 0.05 g Dipotassium phosphate15 g Agar 15 g Water 1000 ml

Colony Form

-   Diameter: 1 to 2 mm-   Color: white-   Shape: circular-   State of elevation: convex-   Margin: entire-   Form of surface: smooth-   Transparency: translucent-   Viscosity: butyrous

1-4. Characteristics Regarding Hydrocarbon Assimilation

The hydrocarbon assimilation of the microorganism of the presentinvention is preferably as shown in Table 2 below.

TABLE 2 Presence/Absence Presence/Absence Hydrocarbon of AssimilationHydrocarbon of Assimilation Glycerol − Salicin + Erythritol −D-cellobiose + D-arabinose − D-maltose + L-arabinose − D-lactose +D-ribose + D-melibiose − D-xylose − D-sucrose + L-xylose − D-trehalose +D-adonitol + D-inulin − Methyl-β-D- − D-melezitose + xylopyranosideD-galactose + D-raffinose − D-glucose + Starch − D-fructose + Glycogen −D-mannose + Xylitol − L-sorbose + Gentiobiose + L-rhamnose −D-turanose + Dulcitol − D-lyxose + Inositol − D-tagatose + D-mannitol +D-fucose − D-sorbitol + L-fucose − Methyl α-D- − D-arabitol −mannopyranoside Methyl α-D- + L-arabitol − glucopyranoside N- +Gluconate + acetylglucosamine Amygdalin + 2- − ketogluconate Arbutin +5- − ketogluconate Esculin Ferric + Citrate

In the table, “+” means positive, and “−” means negative.

1-5. Hepatic Neutral Fat Reducing Activity

The microorganism of the present invention preferably has hepaticneutral fat reducing activity when it is ingested by or administered tohumans or other animals. The neutral fat is not particularly limited,but is generally triglyceride. Since the hepatic neutral fat reducingactivity of the microorganism of the present invention makes it possibleto reduce the amount of neutral fat already accumulated in the liver,the microorganism of the present invention is useful for, for example,treatment or alleviation of humans having fatty liver, as well asprevention of progression of fatty liver in humans with a risk of fattyliver, or health maintenance. Further, the microorganism of the presentinvention is also useful for prevention of progression to NASH, livercirrhosis, liver cancer, and/or heart disease due to hepatic neutral fataccumulation.

The hepatic neutral fat reducing activity of the microorganism of thepresent invention may be measured using a known analysis technique.Specifically, the hepatic neutral fat reducing activity of the presentinvention may be measured using a model animal (for example, mice)according to the procedures below. Mice are fed with the microorganismof the present invention for a certain period of time; thereafter, theirlivers are isolated and neutral fats are extracted according to themethod of Folch et al. (J. Biol. Chem. 1957; 226 (1):497-509). Theamounts of the neutral fats are measured using a kit obtained from acommercial supplier. With the same procedures, the amounts of neutralfats in the livers isolated from mice that have not been fed with themicroorganism of the present invention are measured. Then, the neutralfat amounts in the mice fed with the microorganism of the presentinvention are compared with the neutral fat amounts in the mice unfedwith the microorganism of the present invention, thereby measuring thehepatic neutral fat reducing activity.

1-6. Energy Metabolism Promoting Activity

The microorganism of the present invention preferably has energymetabolism promoting activity in an organism when it is ingested by oradministered to humans or other animals. More specifically, themicroorganism of the present invention has an action of promoting energymetabolism in the intestinal tissue (in particular, in the smallintestinal tissue) and/or liver. By using the microorganism of thepresent invention, it is possible to improve constitutions, or alleviatemetabolic syndrome and/or obesity or the like of humans or other animalsin need of energy metabolism promotion.

As shown in the Examples, the energy metabolism promoting activity ofthe microorganism of the present invention may be confirmed by measuringchanges in expression amounts of mRNA encoding energy metabolism-relatedenzymes (Kondo et al., Am. J. Physiol. Endocrinol Metab, 291,E1092-E1099, 2006). In addition, the energy metabolism promotingactivity of the microorganism of the present invention may be confirmedby calorimetric measurement in humans or other animals before and afterthe intake of the microorganism of the present invention (Sasaki,Measurement of resting energy expenditure and substrates expenditureusing Indirect calorimetry, 24, 5, 1021-1025; Kaiyala et al.,Comparative Biochemistry and Physiology, Part A 158, 252-264, 2011),easurements of oxygen intake amount and carbon dioxide output (Klaus etal., International Journal of Obesity, 29, 615-623, 2005), and/ormeasurements of the activities of metabolism-related enzymes.

1-7. Other Activities

In addition to the above, the microorganism of the present inventionpreferably also has blood glucose level reducing activity, blood neutralfat level reducing activity, blood endotoxin level reducing activity,selective saturated fatty acid reducing activity, fatty acidβ oxidationpromoting activity in the small intestine and the liver, and/or TLR-2mRNA expression promoting activity in the small intestine, and the like.Accordingly, the microorganism of the present invention is useful forthe treatment of diabetes patients based on its blood glucose levelreducing activity, and is also useful for the treatment of septicemiabased on its blood endotoxin level reducing activity. Further, the fattyacid β oxidation promoting activity in the small intestine and the liveris conducive to the energy metabolism promotion mentioned above.Further, since TLR-2m RNA expression in the small intestine increasesthe barrier function of the small-intestinal mucosa, and thereby bringsabout immunostimulatory activity (Cario et al., Gastroenterology, 132,4, 1359-1374, 2007), the effects on the prevention or treatment ofinflammatory bowel disease, bacterial infection, virus infection,endotoxemia, heart disease, atherosclerosis, food allergy, atopicdermatitis, and like are expected.

1-8. Representative Strain

The representative microorganism of the present invention is strainWON0604. This strain was internationally deposited with theInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology (Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki 305-8566 Japan) on Feb. 20, 2012, under accession No. FERMBP-11468. Strain WON0604 satisfies all of the above characteristics 1-1to 1-7. The International Patent Organism Depositary in the NationalInstitute of Advanced Industrial Science and Technology was consolidatedwith the International Patent Organism Depositary in the IncorporatedAdministrative Agency National Institute of Technology and Evaluation(NITE) on April 2012, and their responsibility regarding Patent OrganismDepositary was taken over by the International Patent OrganismDepositary in the Biological Resource Center of the National Instituteof Technology and Evaluation (NITE-IPOD) (Room 120 2-5-8Kazusa-Kamatari, Kisarazu-city, Chiba 292-0818 JAPAN).

The microorganism of the present invention is preferably in an isolatedstate. Further, insofar as the polyamine production promoting activity,the hepatic neutral fat reducing activity and the energy metabolismpromoting activity described above are ensured, the microorganism of thepresent invention may be viable cells or dead cells, and may be in astate of purified cells, roughly purified cells, cells mixed withunpurified medium, or a cell extract. The microorganism of the presentinvention is preferably viable cells because viable cells continuouslyand effectively exhibit polyamine production promoting activity in anorganism. Further, by adding a commonly used freeze-dried preservativeto viable cells to freeze-dry the cells, and conserving the resultingfreeze-dried cells in a refrigerator or in a freezer, it becomespossible to conserve the viable cells for a long period of time. In oneembodiment, the microorganism of the present invention is in afreeze-dried state.

1-9. Method for Obtaining the Microorganism of the Present Invention

The source from which the microorganism of the present invention is tobe isolated is not particularly limited. For example, the microorganismof the present invention may be isolated from foods known to containLactobacillus paracasei therein (for example, various Japanese pickles,Korean pickles, cow's milk, cheese, and the like). Since Lactobacillusparacasei strain WON0604, i.e., the microorganism of the presentinvention, was isolated using Crucian carp sushi as a source, themicroorganism of the present invention may, for example, be isolatedusing Crucian carp sushi as a source. Crucian carp sushi designates afood produced by lactic fermentation of rice and fish produced aroundLake Biwa in Japan. The isolation of lactic acid bacteria from Cruciancarp sushi may be conducted, for example, according to the methoddisclosed in Tuda et al. (Food Sci. Technol. Res., 18 (1), 77-82, 2012).

The isolation of the microorganism of the present invention may beperformed by a known screening method using polyamine productionpromoting activity, hepatic neutral fat reducing activity, and energymetabolism promoting activity described above as indices. For example,the microorganism of the present invention may be isolated by (1)confirming whether the food or the like used as the source has polyamineproduction promoting activity, (2) diluting the source which had itspolyamine production promoting activity confirmed with an appropriatebuffer solution, applying the diluted source to agar medium to culturethe cells into colonies, (3) identifying a colony or colonies havingpolyamine production promoting activity among the colonies formed, (4)extracting 16Sr DNA from the colony, thereby determining the sequencethereof, and thereby judging whether the source belongs to Lactobacillusparacasei.

2. Composition

The composition of the present invention is a composition to which themicroorganism of the present invention has been added. The type and theform of the composition are not particularly limited insofar as thepolyamine production promoting activity and, preferably, the hepaticneutral fat reducing activity and the energy metabolism promotingactivity of the microorganism of the present invention, are notinhibited. However, in view of desirable exhibition of these functionsin animals, in particular, in human organisms, the composition ispreferably a food or beverage composition, or a pharmaceuticalcomposition.

With the addition of the microorganism of the present invention, thecomposition of the present invention has polyamine production promotingactivity, hepatic neutral fat reducing activity, and/or energymetabolism promoting activity. Therefore, in a preferred embodiment, itis possible to use the composition of the present invention for apolyamine production promoter, a hepatic neutral fat reducer, and/or anenergy metabolism promoter. Although the above polyamine productionpromoter and the like may be in the form of a composition, it may alsoconsist only of the microorganism of the present invention.

The composition of the present invention may contain other arbitrarycomponents according to its form and purpose, insofar as the polyamineproduction promoting activity and the like of the microorganism of thepresent invention are not interfered. For example, in view ofmaintaining the growth of the microorganism of the present invention,the composition of the present invention preferably contains, forexample, nutrient compositions suitable for the growth of themicroorganism of the present invention, such as skim milk, dextrin, andthe like.

The amount of the microorganism of the present invention to be added tothe composition of the present invention is not particularly limited,and may be suitably set insofar as the polyamine production promotingactivity and the like of the microorganism are exhibited in animals, inparticular, in human organisms. For example, on a viable cell basis, thecomposition of the present invention may contain the microorganism in anamount of about 1.0×10⁴ to 1.0×10¹⁶ CFU per gram of the composition,preferably 1.0×10⁶ to 1.0×10¹⁴ CFU per gram of the composition, morepreferably 1.0×10⁸ to 1.0×10¹² CFU per gram of the composition.

The type or the form of the food or beverage composition to which themicroorganism of the present invention is added is not particularlylimited, and examples include, in addition to general food and drinks,various functional foods (e.g., food for specified health use, dietarysupplement, supplement, patient food, and health food). By adding themicroorganism of the present invention to such foods, it is possible tofurther improve the polyamine production promoting activity and thelike, and thereby provide an improved food or beverage composition thatmore efficiently promotes polyamine production in an organism. Such animproved food or beverage composition may also be used for homeostaticcontrol in an organism.

When the composition of the present invention is a food or beveragecomposition, the form of the composition is not particularly limitedinsofar as the polyamine production promoting activity and the like ofthe microorganism of the present invention are not inhibited. Examplesof food composition include granules, fine granules, powder, capsules,tablets, gum, jelly, gummy candy, bars, chips, flakes and other generalfoods. Examples of general foods include chocolates, biscuits, candies,cookies, tablet confectioneries, ice cream, sherbet, Udon (wheat)noodles, soba (buckwheat) noodles, pasta, and somen (thin wheat)noodles. Examples of drink compositions include various drinks such aspowder drinks, soft drinks, milk beverages, nutritional beverages,carbonated drinks, and jelly drinks. Examples of the forms of functionalfood include powder, granules, capsules, syrup, tablets, sugar-coatedtablets, and sublingual tablets.

The means for adding the microorganism of the present invention to afood or beverage composition is not particularly limited. For example,the microorganism of the present invention may be added to a food orbeverage composition during the manufacture, processing, or in a finalstep of the food or beverage composition, by way of addition, mixing,infiltration or the like. Further, the microorganism of the presentinvention may be added in the form of powder, granules, capsules, syrup,tablets or the like upon intake of food and drinks.

When the composition of the present invention is a pharmaceuticalcomposition, it is possible to produce various drug forms by mixing themicroorganism of the present invention with a pharmaceuticallyacceptable carrier. The pharmaceutically acceptable carrier is notparticularly limited insofar as the polyamine production promotingactivity and the like of the microorganism of the present invention arenot inhibited. Examples of pharmaceutically acceptable carriers includevarious fillers, expanders, binders, moisturizers, disintegrants,surface active agents, lubricants, and diluents, which are generallyused in the medical field. The form of the pharmaceutical composition inwhich the microorganism of the present invention is added is notparticularly limited. Examples of the form include tablets, pills,powdered drug, liquids, suspensions, emulsions, granules, and capsules.The drug form is preferably a form suitable for oral administration.

By containing the microorganism of the present invention, thepharmaceutical composition of the present invention exhibits polyamineproduction promoting activity as well as, preferably, hepatic neutralfat reducing activity and energy metabolism promoting activity.Therefore, the pharmaceutical composition of the present invention maybe used as a pharmaceutical composition for promoting polyamineproduction in an organism, a pharmaceutical composition for reducinghepatic neutral fat, and/or a pharmaceutical composition for promotingenergy metabolism. Further, based on the hepatic neutral fat reducingactivity of the microorganism of the present invention, thepharmaceutical composition of the present invention may be used as apharmaceutical composition for the prevention or treatment of fattyliver, a pharmaceutical composition for the prevention of progression orthe treatment of NASH, liver cirrhosis, and/or liver cancer.Additionally, based on the energy metabolism promoting activity of themicroorganism of the present invention, the pharmaceutical compositionof the present invention may be used as a pharmaceutical composition forthe prevention or treatment of metabolic syndrome.

The amount of the microorganism of the present invention to be added tothe pharmaceutical composition of the present invention is similar tothe amount defined above for general compositions. The dose of thepharmaceutical composition of the present invention may be suitably setaccording to the symptom, age, weight and the like of the human or theanimal who intakes the composition.

The dosage form of the pharmaceutical composition of the presentinvention is not particularly limited, insofar as the polyamineproduction promoting activity and the like of the microorganism of thepresent invention are exhibited in the body. However, the dosage form ispreferably oral administration.

3. Treatment and Alleviation Method

The microorganism of the present invention has polyamine productionpromoting activity in an organism of animals, in particular, humans.Polyamine is known to have an anti-aging effect, arteriosclerosisprogression retardation activity, acute and chronic inflammationsuppressing activity, neutral fat reducing activity, insulin resistancealleviating activity, anti-obesity activity, cholesterol leveldecreasing activity, basal metabolism increasing activity, antiallergicactivity, immunostimulatory activity, and the like. Therefore, a methodfor preventing, treating, or alleviating various diseases using theseactivities, comprising administering the microorganism of the presentinvention, may be provided. Further, in a preferred embodiment, themicroorganism of the present invention has hepatic neutral fat reducingactivity. Therefore, by administering the microorganism of the presentinvention to a human or an animal in need of reduction in hepaticneutral fat, it is possible to reduce the hepatic neutral fat. Further,in a preferred embodiment, the present invention has energy metabolismpromoting activity in an organism. Therefore, by administering themicroorganism of the present invention to a human or an animal in needof promotion of energy metabolism, it is possible to suppress bodyweight gain, suppress obesity, reduce body fat, reduce visceral fat, andprevent, alleviate, or treat metabolic syndrome.

The dose of the microorganism of the present invention, or the dose ofthe pharmaceutical composition of the present invention in which themicroorganism of the present invention is added in order to carry outthe above method may be suitably set according to the symptom or thelike of the patient. For example, the dose is 1.0×10⁵ to 1.0 to 10¹⁵CFU/kg/day.

EXAMPLES Example 1 Isolation of Microorganism

0.8 to 1.0 g of homogenized Crucian carp sushi was placed in a 15 mlcentrifuge tube, and ten times the amount of physiological salinesolution was added and stirred. 1 ml of the stirred suspension wasremoved and placed in a 15 ml centrifuge tube together with 9 ml of aphysiological saline solution, followed by stepwise dilution. 100 μl ofeach diluent was obtained, inoculated into MRS agar medium, and culturedfor 24 hours at 37° C. under an anaerobic condition. After the culture,the cells were purified until they became a single colony. Theseoperations were performed as aseptic manipulation.

The isolated strain WON0604 was deposited with the International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology (1-1-1 Higashi, Tsukuba-city, Ibaraki 305-8566 Japan),under accession No. FERM BP-11468.

EXAMPLE 2 Polyamine Production Promoting Activity

KK-Ay mice (type II diabetes model, male) at 4 weeks of age werefastened for 16 hours, and blood was drawn from a single capillaryvessel in the tail vein (about 75 pl). Plasma was prepared bycentrifugation (12,000 rpm (15,000×g)×5 minutes), and the blood glucoseconcentration and the blood neutral fat concentration were measured.Mice with no abnormal health conditions were classified into a controlgroup and a test group, while eliminating bias in body weight (the daybefore the grouping) and blood glucose concentration by way ofstratified random sampling using SAS software (R9.1, SAS InstituteJapan). For 28 days, the control group was freely fed with a controlmeal (AIN-93G), and the test group was freely fed with a test meal mixedwith strain WON0604 at a proportion of 1.0×10⁹ CFU/day (viable cells).Thereafter, the cecum and the small intestine tissue were dissected, andthe polyamine concentrations in the cecal content and the smallintestine tissue were measured. Table 3 shows the values of each testgroup relative to the polyamine concentration of the control group,which is assumed as 1. n=3 both for the cecal content and the smallintestine tissue. The polyamine concentration in the cecal content wasmeasured by an on-column derivatization method using O-phthalaldehyde.The polyamine concentration in the small intestine tissue was measuredusing a CE-TOFMS system (Agilent Technologies). The significance testwas performed according to Welch's t-test.

TABLE 3 Polyamine amount in small intestine Relative value againstcontrol P Value Putrescine 1.4 0.007* Spermidine 1.3 0.064  Spermine 1.10.523  *P < 0.01

TABLE 4 Polyamine amount in cecum Relative value against control P ValuePutrescine 0.79 0.412 Spermidine 1.07 0.549 Spermine 0.93 0.549

As shown in Table 4, the polyamine concentration in the cecum was notincreased by the intake of strain WON0604. In contrast, as shown inTable 3, putrescine was significantly increased in the small intestine,and there was also a tendency for spermidine and spermine to increase inthe small intestine. The results thus revealed that polyamine in thesmall intestine was increased by the intake of strain WON0604. Thisclarified that the microorganism of the present invention has an actionof promoting polyamine synthesis capability itself in the smallintestine (i.e., polyamine synthesis promoting activity).

EXAMPLE 3 Energy Metabolism Promoting Activity

Similarly to Example 2, KK-Ay mice (type II diabetes model, male) at 4weeks of age were classified into a control group and a test group; thecontrol group was freely fed with a control meal (AIN-93G), and the testgroup was freely fed with a test meal mixed with strain WON0604 at aproportion of 1.0×10⁹ CFU/day (viable cells) for 28 days. Thereafter,mRNA expression levels of various energy metabolism-related enzymes inthe liver and the small intestine were measured. The mRNA extraction andpurification were performed using a RiboPure® Kit (Ambion). Table 5shows the results, i.e., the values of the test meal groups relative tothe value of the control group, which is assumed as 1. In this test,n=13, and the relative values were calculated using average values.

TABLE 5 Relative mRNA expression level in liver P Value Acox1 1.160.020* Ucp2 1.33 0.048* Acot2 1.29 0.058  *P < 0.05

TABLE 6 Relative mRNA expression level in small intestine P Value Acox11.31 0.001* Ucp2 1.17 0.067  Acot2 1.21 0.066  *P < 0.05

The results shown in Tables 5 and 6 confirmed that the intake of themicroorganism of the present invention increased the mRNA expressionamounts of Acoxl, Ucp2 and Acot2 in the liver and the small intestine.The relevance of the expression of these genes and energy metabolismpromotion has been reported by Kondo et al. (Kondo H, et al., Am JPhysiol. Endocrinol Metab 2006; 291(5): E1092-9). The results confirmedthat, in addition to the polyamine production promoting effect, thepossibility of promotion in energy metabolism in the liver and the smallintestine by the intake of the microorganism of the present inventionwas also confirmed.

EXAMPLE 4 Hepatic Neutral Fat Reducing Activity

Similarly to Example 2, KK-Ay mice (type II diabetes model, male) at 4weeks of age were classified into a control group and a test group; thecontrol group was freely fed with a control meal (AIN-93G) for 28 days,and each test group was freely fed with a test meal mixed with 3% of atest meal containing strain WON0604, strain WON1052, strain WON1081, orstrain WON1033 at a proportion of 1.0×10⁹ CFU/day (viable cells) for 28days. These lactic acid bacterial strains other than strain WON0604 wereobtained by selecting strains resistant to gastric acid and biliary acidthrough an in vitro test, and then further selecting strains withpotential efficacies with respect to blood neutral fat and hepaticneutral fat through an exploratory animal test using KK-Ay mice.Thereafter, the liver was removed from each mouse in a fasted condition.The outer region of the lobus hepatis sinister was weighed, and thelipid fraction was extracted using the method of Folch et al. (Folch J.et al., J. Biol. Chem. 1957; 226(1):497-509) and solubilized usingisopropanol; thereafter, the neutral fat concentration was measuredusing a measurement kit obtained from a commercial supplier(Triglyceride E-test Wako; Wako Pure Chemical Industries, Ltd.). Table 7shows average values of the measurement results. Each test group hadeight mice. The significance test was performed according to t-test.

As shown in Table 7, the hepatic TG concentration was decreased only inthe mice fed with strain WON0604, compared with the control group withsignificant difference; and the hepatic TG concentration reducingactivity was not observed in the comparative test groups fed with lacticacid bacteria other than strain WON0604. The results thus confirmed thepossibility of hepatic neutral fat reducing activity by themicroorganism of the present invention, in addition to the polyamineproduction promoting effect.

TABLE 7 Hepatic TG (mg/g liver) Control group 29.5 ± 8.5 WON0604  18.4 ±6.8* WON1052 26.4 ± 6.3 WON1081 34.6 ± 9.0 WON1033 25.3 ± 9.7 *P < 0.05

Accession Number

-   FERM BP-11468

1. Lactobacillus paracasei strain WON0604 (FERN BP-11468).
 2. Acomposition to which Lactobacillus paracasei strain WON0604 according toclaim 1 has been added.